Different techniques can be used to characterize pathology samples such as tumors. One of such techniques investigates homogenized samples and determines information from the homogenized sample, e.g., within a test tube, and the other collects spatially orientated information.
Homogenized tissue sample tests can test for different characteristics—however, the whole contents of the test tube is averaged for the test. Tests of these types include polymerase chain reaction or PCR, Western blotting that can be used to quantify concentrations of types of proteins in a sample, DNA arrays, that can be used to quantify the amount of DNA in specified sequences, RNA arrays that can be used to quantify specific sequences of messenger RNA and thus determine the expression level of many different genes, and others. These kinds of tests can be very specific—for example, quantitative PCR can be used to determine the relative level of sequences that differ by only a single mutation. However, the specificity is reduced since the test is measuring over the entire sample.
Staining techniques can also be used. In slide based tests the sample is sectioned into thin sections (typically 5 microns) and placed on a microscope slide for observation with a microscope, photo microscopy or image analysis. Stains are used on the tissue to make certain features visible. One stain is the classic H&E stain. This stain allows pathologists to view the overall morphology of a tissue and identify areas of tumor based on morphological features that show up under the stain. Other stain techniques produce other results. For example, an IHC (Immunohistochemistry) reagent is a custom antibody to a given target which can be a protein or other chemical linked (either before or after it attaches to its target) to a stain that will be visible in the microscope. This marker might be an enzyme which will catalyze a color generating reaction. IHC can be used to visualize contents of a slide to determine that a target molecule is present. IHC can also be used to specify the specific cells or even sub cellular structures in which the target molecule is present. By using several distinguishable stains linked to different antibodies, it is possible to characterize the extent of co-localization of several targets or determine that they are found in different cells or organelles.
FISH (Fluorescent in-situ hybridization) can locate the position on a chromosome of a given genetic sequence by using several probes linked to different colored dyes. Fish makes it possible to see the spatial relationship of different loci. For instance FISH can be used to detect chromosome translocation that cause leukemia by marking two loci known to be brought together by a translocation that causes leukemia with red and green fluorochromes. If the translocation has occurred, these 2 probes will be brought next to each other and will appear to be a single yellow dot. Thus FISH can produce detailed spatial data on the location of gene sequences.
In general, the operation on homogenized tissue make many measurements of different targets but are in effect averaging over the entire block that was homogenized. This has limited their utility in practical diagnosis because the tissue sample delivered to a pathologist is rarely entirely cancer. In cancer surgery, the goal is to remove tissue until the margins are clear (that is free of cancer). In biopsy samples, there is often no way to assure that only tumor is sampled. Further, it is known that many cancers are themselves genetically and metabolically heterogeneous. This means that the numbers produced by all of the homogenization methods may be averages of tumor and non tumor tissue or different regions of the tumor.
Slide based methods overcome the localization limitations but suffer from restrictions on the number of chemical species that can be detected at once. While DNA arrays can test for thousands of sequences at once, FISH is restricted to 4 or 5 probes at a time. Similarly IHC is limited by the number of stains that can be attached and visualized, e.g., 3 or 4.
These limitations mean that in practice it is possible to know the amount of various types of DNA, RNA and protein in a tissue in detail but not the precise location of those species or it is possible to know the location of a few species with high spatial accurately.
Other techniques collect spatially oriented information from slides. Laser capture microdissection, for example, uses a laser to release a chosen section of the tissue, e.g. while observing the tissue on a slide under a microscope (with no cover slip). The released sample is captured in a vial, and the localized sample is tested using one of the homogenization methods mentioned above. This allows obtaining test information for a known location. While this is difficult at the sub cellular level, it is possible to select one or a few cells that are known to be part of a tumor.
Laser capture microdissection has not been widely used because of its disadvantages. One is the cost and complexity of the equipment involved. Also, since the sample is collected after it has been fixed and possibly stained these processes may disrupt the target molecules and prevent some chemical methods from operating correctly. Also, since the size of the area sampled is inherently limited by the collection technique, it may be too small relative to the tumor that is to be characterized.